The transfer of genetic material into cells in mammals is of increasing commercial importance. For instance, gene therapy procedures are used to correct acquired and inherited genetic defects, cancer, and viral infection. The ability to express artificial genes in humans facilitates the prevention and/or cure of many important human diseases, including many diseases which are not amenable to treatment by other therapies.
One of the most common fatal genetic diseases in humans is cystic fibrosis (CF). Cystic fibrosis (CF), a spectrum of exocrine tissue dysfunction,.which eventually leads to respiratory failure and death results from a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene has now been localized to chromosome 7q31, and cloned. A 3 bp deletion, resulting in the loss of a phenylalanine residue at amino acid position 508, is present in approximately 70% of CF chromosomes, but is not seen on normal chromosomes. The other 30% of CF mutations are heterogenous and include deletion, missense, and splice-site mutations.
The major mortality in CF is from pulmonary disease. There is no cure for CF. Current treatments for CF include antibiotics to treat bacteria (pseudomonas) infection, and respiratory physical therapies to remove the mucus blocking the airways of the patients. A recent therapy for CF is recombinant Dnase ("THERAZYME") developed by Genentech to reduce the thickness of the mucus. The drug is expensive and requires daily dose inhalations and combination with physical therapy to remove the mucus from the lungs of the patients. It does not treat the basic defect of CF, that is, the malfunction in chloride transport.
Gene therapy has the potential to provide long term relief in patients by expressing the normal gene in the diseased cells to form normal chloride channel function. Transfection of even a single normal copy of a functional CFTR gene abolishes the CF secretory defect in CF cell lines, an observation which supports the feasibility of gene therapy for CF. see, e.g., Capelen et al. Nature Medicine 1(1): 39 (1995), McLachlan et al., Gene Ther. 3(12): 1113-23 (1996).
Several approaches for introducing functional new genetic material into cells in vivo have been used. These include liposome based gene delivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414) and replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome (see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990); Kolberg (1992) J. NIH Res. 4:43, and Cometta et al. Hum. Gene Ther. 2:215 (1991)). Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof See, e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991); Wong-Staal et al., PCT/IJS94/05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and the references therein, and Yu et al., Gene Therapy (1994) supra). For a review of gene therapy procedures, see Anderson, Science (1992) 256:808-813; Nabel and Felgner (1993) TIBTECH 11: 211-217; Mitani and Caskey (1993) TIBTECH 11: 162-166; Mulligan (1993) Science 926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1) 31-44; Haddada et al. (1995) in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) Springer-Verlag, Heidelberg Germany; and Yu et al., Gene Therapy (1994) 1:13-26.
AAV-based vectors are used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for an overview of AAV vectors. Construction of recombinant AAV vectors are described in a number of publications, including Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol., 63:03822-3828. Cell lines that can be transduced by AAV include those described in Lebkowski et al. (1988) Mol. Cell. Biol., 8:3988-3996.
AAV has also been used to deliver CFTR genes in vivo Flotte, T. et al., Hum. Gene Ther. 7:1145-59 (1996); Flotte, T. R. et al., Proc. Natl. Acad. Sci. USA 90:10613-7 (1993); Flotte, T. R. et al. J. Biol. Chem. 268:3781-90 (1993); and Flotte, T. R. et al., Adv. Pharmacol. 40:85-101 (1997).
Currently, three vector systems are being tested to deliver CFTR gene into airway epithelial cells, a non-viral vector (liposomes), and two viral vectors, adenovirus and AAV. Recent clinical trials have demonstrated that the AAV vector can efficiently and persistently transfer the CFTR gene into the airway epithelimn of patients without any adverse effects (see, Flotte, T. et al., Hum. Gene Ther. supra). In spite of these advantages, previous AAV vectors do not express sufficient levels of CFTR. Because of size constraints, an effective promoter cannot be accommodated with the CFTR gene. This limits the clinical efficacy of the vector for gene therapy of cystic fibrosis. The present invention addresses these and other needs.